Sample preparation and data collection for serial block face scanning electron microscopy of mammalian cell monolayers

Volume electron microscopy encompasses a set of electron microscopy techniques that can be used to examine the ultrastructure of biological tissues and cells in three dimensions. Two block face techniques, focused ion beam scanning electron microscopy (FIB-SEM) and serial block face scanning electron microscopy (SBF-SEM) have often been used to study biological tissue samples. More recently, these techniques have been adapted to in vitro tissue culture samples. Here we describe step-by-step protocols for two sample embedding methods for in vitro tissue culture cells intended to be studied using SBF-SEM. The first focuses on cell pellet embedding and the second on en face embedding. En face embedding can be combined with light microscopy, and this CLEM workflow can be used to identify specific biological events by light microscopy, which can then be imaged using SBF-SEM. We systematically outline the steps necessary to fix, stain, embed and image adherent tissue culture cell monolayers by SBF-SEM. In addition to sample preparation, we discuss optimization of parameters for data collection. We highlight the challenges and key steps of sample preparation, and the consideration of imaging variables.


Attachments
Guidelines TIMING This protocol can be performed over multiple days.Once samples are embedded in resin they are stable at room temperature inde nitely once in ltrated by and polymerized in resin.
Step 1: Culturing mammalian cells, plus an additional 24 -48 h for cells to grow Step 2: Bright eld or uorescence microscopy for CLEM work ow with en face samples, 1 -2 h Steps 3-5: 2 h xation at room temperature plus an additional 24 h at 4 °C.
Step 6-19: Sample staining, embedding and polymerization, 5 days plus any additional time for reagent setup and clean up.
Step 20-28: Cell pellet sample trimming and mounting, 2 h plus any additional time for epoxy polymerization, reagent setup and clean up.
Steps 29-47: en face sample trimming and mounting, 4 h plus any additional time for epoxy polymerization, reagent setup and clean up.
Steps 48-49: sample loading on the SBF-SEM, 1 h plus an additional 12 h to let the system equilibrate.
Steps 50-62: sample imaging, several hours to days depending on the number of ROIs being collected, the volume assigned as well as pixel size and dwell time.

Safety warnings
Caution steps in the protocol have been indicated.Please refer to the material safety data sheet of the compound to ensure its careful handling.Culture adherent mammalian cells for 24 -48 h in a 60 mm or 100 mm tissue culture dish for cell pellet samples, or 35 mm tissue culture dish with a gridded glass bottom for CLEM samples ( nal density 3-6 x 10 5 cells) according to the standard protocol for the cell line.In our experiments, Vero cells were cultured in complete DMEM:HG media.

1.1
Note: For cell pellet samples, the cells can be collected when they reach 90% con uence.
This ensures that there are enough cells to form a large cell pellet.For CLEM, the cells should be at 70% con uence or less, as this will allow you to track the position of a cell of interest by both light and electron microscopy.

2
For en face embedding of cells, proceed with light microscopy to identify speci c cells of interest.Depending on the biological event being studied, you can use bright eld or uorescence microscopy to identify cells of interest (Figure 1A).For cell pellet samples, proceed directly to step 4.

2.1
Critical step: 1) Image cells in an environmental chamber where temperature, humidity and CO 2 concentrations are maintained 2) Capture images at high magni cation (40X or 60X) and low magni cation (5X or 10X) of multiple regions of interest using DIC and uorescence imaging, as appropriate, to identify cells of interest.Images collected at high magni cation will allow you to record both the cell shape and a more precise position relative to the surrounding cells.From images collected at low magni cation, a map of the cell location showing the gridded lines, numbers or letters can be used as a marker when trimming the EM sample block, and searching the target cell of interest under EM.We recommend selecting 2 -4 regions of interest per sample, that are located far apart from each other on the dish.This ensures that each region of interest will be reliably recovered during the subsequent processing steps when the sample is physically cut away from the polymerized resin.

3
Discard tissue culture media, and gently pipette 2 mL of xative (2% PFA and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2)) to x the cells.For cell lines that are more sensitive, add 1 mL of pre-warmed 2x xative (4% PFA and 5% glutaraldehyde in 0.2M sodium cacodylate buffer pH 7.2) directly into the dish with 1 mL of culture media.

3.1
Critical step: xative solution should be freshly prepared

4.1
Critical step: 1) Scrape cells in a single direction, such that you observe a sheet of cells coming off the dish.2) In-dish xation should be limited to less than 3 mins to avoid disintegration of the cell monolayer which can make recovery of the dense cell pellet during centrifugation tough.3) For cell pellet samples, avoid centrifugation moving forward and pellet samples by gravity.

5
Replace with 1 mL fresh xative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2)) and continue to x the cells overnight at 4 °C.

6
Discard xative and add 1 mL 0.1 M cacodylate buffer for 10 min at room temperature.Repeat this buffer rinsing step two more times.

7
Discard remaining buffer and incubate the sample in freshly made 200 µL reduced osmium solution that contains 2% osmium, 1.5% potassium ferrocyanide in 0.1 M sodium cacodylate buffer, for 1.5 h at room temperature in the dark.

7.1
Caution: do not disturb the cell pellet during all sample processing steps.

8
Wash the sample in 1 mL ddH 2 O for 3 min at room temperature.Repeat this wash step four more times.

10
Wash the sample in 1 mL ddH 2 O for 3 min at room temperature.Repeat this wash step four more times.

11
Incubate the sample in 200 µL 2% Osmium in ddH2O for 40 min at room temperature.
12 Wash the sample in 1 mL ddH 2 O for 3 min at room temperature.Repeat this wash step four more times.

13
IIncubate the sample in 0. Durcupan, which is easier for sectioning with an ultramicrotome.However, EMbed [2] will dissolve the cell culture dish, therefore it is not appropriate for an en face CLEM project.

19
For sample embedding of en face samples, tilt the dish at a 45 ° angle in order to discard as much leftover resin from the dish as possible.Add a thin layer of 100% Durcupan resin (~100 µl) directly onto the glass coverslip.For cell pellet samples, there are two options when sample embedding: 1) use a wood applicator stick to transfer the sample to an embedding mold, or 2) directly embed the sample inside the tube if the cell pellet is very small.With either sample embedding method, add 100% Durcupan to ll the embedding mold or ~200 µL to the tube if directly embedding the sample inside the tube.Place embedded samples in a 60 °C oven for 48 -60 h to allow resin-in ltrated samples to polymerize and harden.19.1 Critical step: 1) for en face samples, be sure to add a requisite volume of 100% Durcupan (one or two drops) that will cover just the glass coverslip and not the surrounding areas of the dish and 2) for cell pellet samples in which the sample is very small, sample embedding directly in the tube is recommended.

19.2
Note: Annotate samples clearly to prevent any confusion downstream when working with multiple samples.For en face samples, write down a sample number on a piece of paper and place it on the plastic area of the dish, so that any leftover resin will stick the number tightly to the dish.For cell pellet samples, put a sample number into an embedding mold, preventing overlap with the sample; alternatively, stick it to the inner wall of the tube.

20
Bend the sample embedding mold, or cut open a tube using a razor blade to take out a polymerized sample block.

21
Lock the sample block on the specimen holder of the microtome, then trim the sample block under the microtome.Trim one edge of the sample block containing the cell pellet using a razor blade to expose the cell sample.

22
Place the sample block in the specimen arc of the ultramicrotome and section the exposed sample surface using a Diatome Histo Diamond knife to create the blockface.The knife holder and specimen arc angles should be set to zero.

23
Take the sample block out of the specimen arc.Using a razor blade, trim the remaining edges of the sample block into a pillar.
23.1 Critical step: While trimming the sample block edges, ensure that: 1) the excess resin is trimmed away on the remaining edges in order to expose the sample, 2) each side of the pillar is of an equal dimension relative to the block face and 3) Typically, samples are trimmed into a pyramid shaped block that contains no regions of empty resin i.e. some of the sample is exposed on every side [3-5].Samples can also be trimmed to a cube/ cuboid shape, ~0.5 mm x 0.5 mm and of a similar height.

24
Place the sample block under a stereomicroscope and cut away the newly created 3View sample blockface pillar with a razor blade.

24.1
Note: Laying the block at and protecting the pillar with scotch tape before cutting will help to ensure that the pillar is not lost.Doing so on a piece of white paper will contrast the osmium-darkened sample, making the pillar easier to see.
Sample trimming and mounting: cell pellet samples 25.1 Critical step: While gluing the sample, ensure that 1) the sample block face created using a Diatome Histo Diamond knife is glued directly to the surface of the 3View SEM pin stub, with the side cut away from the sample block facing up and 2) each side of the pillar is also covered by silver epoxy.

26
Place the 3View pin stub in a 60 °C oven overnight to polymerize the epoxy.

27
Place the pin stub in the specimen arc of the ultramicrotome and section the exposed sample surface using a Diatome Histo Diamond knife, where both the knife holder and specimen arc are set to angles of zero.

28
Remove the pin stub from the ultramicrotome and trim away any excess epoxy from the sides of the pillar.

29
Freeze the dish with the resin embedded sample in liquid nitrogen to carefully separate the glass coverslip from the polymerized cell monolayer.Repeat these freeze thaw cycles if necessary.

29.1
Critical step: Immediately after freezing, use a very sharp razor blade to separate the perimeter of the circular coverslip from the dish, by lodging the razor edge between the perimeter of the coverslip and dish while rotating the dish.

30
Identify the region of interest (ROI) and its respective, letter-number location square within the location-based grid on the sample side of the polymerized monolayer (Figure 1B).

31
On the sample side mark off a square around the letter-number location containing the ROI, using a razor blade (Figure 1C).

31.1
Critical step: Take care not to damage the surface of the monolayer-ROI during this and all subsequent steps, as it is no longer protected by the glass coverslip; simply touching it may destroy the sample and/or its respective location-speci c letter/number.

32
Turn over the polymerized sample monolayer and mark the location of the ROI with an "X" on the non-sample side of the resin; use the square marked by a razor on the sample side as a reference in doing this (Figure 1C).

33
Using a stereomicroscope, con rm that the "X" on the non-sample side aligns with the ROI on the sample side (Figure 1C).

34
Carefully place a 1 x 2 mm slot grid over the ROI on the sample side.Ensure that the ROI is centered within the slot.Using a razor blade, place marks at the sides of the slot grid so that the marks are parallel to the 2 mm side of the slot.These marks, in combination with the marks made in step 31, should create a rectangle equal to the diameter of the slot grid (Figure 1C).

35
Using a razor separate this rectangle, one side at a time, from the larger polymerized monolayer (Figure 1C).

35.1
Critical step: Con rm that the non-sample side of the rectangular resin piece contains the centered "X" from step 30 (Figure 1C).

36
Set the rectangular resin piece aside, either in a microcentrifuge tube or a small gelatin capsule, so as not to lose the sample during subsequent steps.

37
Using a conductive silver epoxy, glue two slot grids one on top of the other to the top of a 3View SEM pin stub (EMS Cat# 75959-03) (Figure 1C).

37.1
Note: Make sure that the slots of both slot grids are aligned

38
Place the 3View SEM slot grid-pin stub into a 60°C oven overnight to polymerize the epoxy.

38.1
Note: Once dried, remove the now polymerized, excess silver epoxy from the interior of the superimposed slots using the edge of a razor or scalpel.

39
Glue the ends of the rectangular resin piece to the broader parts of the slot grid, sample side down, using the same conductive epoxy.
39.1 Critical step: Ensure that the ROI target is centered in the slot of the slot grid and that the "X" marked on the non-sample side in Step 30 faces up.Be very careful to avoid getting epoxy on the ROI itself by placing a piece of tape on the sample side.

40
Allow the glue between the 3View SEM-slot grid pin stub and the rectangular resin piece to fully polymerize.Leave samples in a 60 °C oven overnight.

41
Place the 3View SEM slot grid-pin stub into the specimen arc of the ultramicrotome, taking care to ensure that both the specimen arc and knife holder are at zero degrees.

42
Using a Diatome Histo Diamond knife, section the now exposed non-sample side, at 500 nm section thickness and speed 1 mm/second.Continue to section until most of the resin from the non-sample side is removed and the rectangle is as thin as possible.